Quantification of biomolecules responsible for biomarkers in the surface-enhanced Raman spectra of bacteria using liquid chromatography-mass spectrometry.

Recently, specific biomarkers in the surface-enhanced Raman scattering (SERS) spectra of bacteria have been successfully exploited for rapid bacterial antibiotic susceptibility testing (AST) - dubbed SERS-AST. The biomolecules responsible for these bacterial SERS biomarkers have been identified as several purine derivative metabolites involved in bacterial purine salvage pathways (W. R. Premasiri, J. C. Lee, A. Sauer-Budge, R. Theberge, C. E. Costello and L. D. Ziegler, Anal. Bioanal. Chem., 2016, 408, 4631). Here we quantified these metabolites in the SERS spectra of Staphylococcus aureus and Escherichia coli using ultra-performance liquid chromatography/electrospray ionization-mass spectrometry (UPLC/ESI-MS). The time dependences of the concentrations of these molecules were measured using 13C- or 12C-purine derivatives as internal and external standards respectively in UPLC/ESI-MS measurements. Surprisingly, a single S. aureus and an E. coli cell were found to release millions of adenine and hypoxanthine into a water environment in an hour respectively. Furthermore, simulated SERS spectra of bacterial supernatants based on the mixtures of purine derivatives with measured concentrations also show great similarity with those of the corresponding bacterial samples. Our results not only provide a quantitative foundation for the emerging SERS-AST method but also suggest the potential of exploiting SERS for in situ monitoring the changes in bacterial purine salvage processes in response to different physical and chemical challenges.

On-chip thin film Zernike phase plate for in-focus transmission electron microscopy imaging of organic materials.

Transmission electron microscopy (TEM) is a powerful tool for imaging nanostructures, yet its capability is limited with respect to the imaging of organic materials because of the intrinsic low contrast problem. TEM phase plates have been in development for decades, yet a reliable phase plate technique has not been available because the performance of TEM phase plates deteriorates too quickly. Such an obstacle prohibits in-focus TEM phase imaging to be routinely achievable, thus limiting the technique being used in practical applications. Here we present an on-chip thin film Zernike phase plate which can effectively release charging and allow reliable in-focus TEM images of organic materials with enhanced contrast to be routinely obtained. With this stable system, we were able to characterize many polymer solar cell specimens and consequently identified and verified the existence of an unexpected nanoparticle phase. Furthermore, we were also able to observe the fine structures of an Escherichia coli specimen, without staining, using this on-chip thin film phase plate. Our system, which can be installed on a commercial TEM, opens up exciting possibilities for TEM to characterize organic materials.

Transparent Raman-enhancing substrates for microbiological monitoring and in situ pollutant detection.

Opaque Raman-enhancing substrates made of Ag nanoparticles on incompletely oxidized aluminum templates have been rendered transparent by an ion-drift process to complete the oxidation. The result shows that the transparent substrates exhibit high/uniform surface-enhanced Raman scattering (SERS) capability and good optical transmissivity, allowing for concurrent SERS characterization and high contrast transmission-mode optical imaging of S. aureus bacteria. We also demonstrate that the transparent substrates can used in conjunction with optical fibers as SERS sensors for in situ detection of malachite green down to 10(-9) M.